1. Field of the Invention
The present invention relates generally to a method of a tailored multi-site combinatorial assembly (“TMCA”) as a method of producing a plurality of progeny polynucleotides and making specific changes to a gene and reassembling mutations or changes at multiple sites. The mutations or changes are designed and synthesized on short oligonucleotides. The oligonucleotides are annealed to a template DNA comprising the wild type gene. A DNA polymerase is used to amplify the whole DNA. The resulting amplified DNA is recovered from a host. The advantages of this method are speed, technical simplicity, and ability to control the assembly.
2. Description of the Background
Published methods of making changes to a gene use, for example, an error-prone PCR, Invitrogen's Gene Tailor site-directed Mutagenesis Kit™, Stratagene's QuickChange Mutagenesis Kit™, overlap PCR and PCR-based ligation/recombination. A survey of the known methods reveals that these methods tend to face a primary difficulty of generating a mutation and/or modification at a single site/neighborhood region and/or are laborious for making modifications at multiple regions.
U.S. Pat. No. 7,202,086 (“the '086 patent”) claims a process for mutagenesis using at least 5 oligonucleotides overlapping or not and dsDNA (plasmid) to generate a library of mutated genes, wherein each mutation is present on average in less then ⅕ of the genes in the library. The '086 patent describes that the disclosed invention is different from that of the prior art because the '086 patent requires controlling the frequency of mutations to avoid “excess of mutations” in one DNA molecule (col. 5, lines 28-45). It is desired to get mutants each containing one mutation. In order to achieve this goal, the ratio between the quantity of each mutant oligonucleotide and the quantity of a template must be between 0.01-100 (col. 5, lines 28-45). This feature is distinguished from that of the prior art, wherein using several oligonucleotides simultaneously leads to the level of incorporation of each primer of more than 75% (col. 5, lines 28-45). The '086 patent requires controlling the frequency of mutations to avoid “excess of mutations” in one DNA molecule and to generate mutants each containing one mutation.
U.S. Pat. No. 7,132,265 (“the '265 patent”) and U.S. patent publication 2003/0064516 claim a method of introducing mutations into a single stranded DNA (“ssDNA”) molecule comprising annealing a primer, synthesizing a DNA strand, and digesting the DNA molecule. The TCMA method uses double stranded DNA (“dsDNA”) as the template. The '265 patent makes a clear distinction between using ssDNA and dsDNA as the starting substrate for mutagenesis protocols (see, e.g., col. 6, lines 45-55).
U.S. published application 2003/0194807 are directed to a library wherein mutants of a protein comprise a single predetermined amino acid in one or more positions in a defined region, wherein the defined region is at least three amino acids. Only a single change at a location is permitted, i.e., excludes degenerative changes at a particular amino acid position.
Each of U.S. published applications 2006/0051748; 2006/0134624; 2004/0248131; and 2002/0083488; and U.S. Pat. Nos. 6,673,610; 6,335,160; and 5,354,670 require ligating synthesized DNA to produce progeny circular DNA with the mutations.
Further, U.S. published application 2006/0051748 requires using a flap endonuclease and annealing all primers to the same DNA strand. U.S. published application 2006/0134624 requires using two primers consecutively (i.e., not in one reaction). In U.S. published application 2004/0248131, primers are annealed to two strands, wherein the primers have to comprise 2-4 complementary base pairs. U.S. Pat. No. 6,673,610 and U.S. published application 2002/0083488 require using fragments produced by digesting of the parent DNA strand as a megaprimer for obtaining a circular DNA used in transformation. U.S. Pat. No. 6,335,160 is directed to the gene assembly from overlapping fragments and generating a recombinant library. Finally, U.S. Pat. No. 5,354,670 requires two transformation steps and an intermediate treatment with a restriction.
Each of U.S. Pat. Nos. 7,176,004; 6,713,285; 6,391,548; and 5,932,419; and U.S. published applications 20040253729 and 20030032037 require two primers to anneal to two different strands for initiating amplification in opposite directions (i.e., forward and reverse primers) and to have complementary regions. U.S. Pat. Nos. 7,078,389 and 5,935,830 require a primer to comprise a mutagen (e.g., psoralen) that interacts with a template so that a triple-stranded molecule is formed.
U.S. published application 2006/0228786 require conducting polymerization of two strands using two different primers in two different reaction followed by annealing of the synthesized ssDNA molecules. U.S. published application 2003/0077613 are directed to a method for gene assembly and creating a library, wherein an assembled gene (ssDNA) is annealed with a scaffold DNA to fill gaps and generated dsDNA which is subcloned into a vector.
U.S published application 2004/0002057 describes a method for detecting a ligand in a sample which does not comprise mutagenesis. U.S. published application 2004/0002057 describes a method of establishing a mutant E. coli strain by using a mutagen in cultured cells. U.S. published application 2006/0199222 describes a generic method of directed evolution wherein mutated DNA is transformed into a particular, Bacillus strain.
There still exists a need for better and more effective method of generating a specific gene variants and a combinatorial gene library efficiently and quickly.